Fuels, plastics, and chemicals derived from agricultural feedstocks are receiving considerable attention as the world looks for solutions to dwindling non-renewable petroleum resources (Herrera, (2006), Nature Biotechnol. 24:755-760; Kanun et al., (2007), Adv. Biochem. Eng. Biotechnol. 105:175-204; Ragauskas et al., (2006), Science 311:484-489). In the United States, efforts have primarily focused on biofuels such as ethanol produced from the starch in maize kernels. This feedstock will likely be replaced by lignocellulosic biomass since U.S. maize production capacity can only supply a portion of the feedstock required for the widespread production of ethanol (DOE (2006), DOE/SC-0095, U.S. Department of Energy Office of Science and Office of Energy Efficiency and Renewable Energy www.doegenomestolife.org/biofuels/); Service, R. F. (2007) Science 315:1488-1491). Current technologies for conversion of lignocellulosic biomass to biofuels are hindered by high costs and a significant amount of research effort is underway to create more efficient, less expensive processes (Service, R. F. (2007) Science 315:1488-1491). Engineering bioenergy crops to synthesize industrial materials would provide better economics for both the fuel and co-product components.
Polyhydroxyalkanoates (PHAs), a family of naturally renewable and biodegradable plastics, fit nicely into a biorefinery concept (Kamm et al., (2007), Adv. Biochem. Eng. Biotechnol. 105:175-204; Ragauskas et al., (2006), Science 311:484-489) as a value added co-product to lignocellulosic derived biofuels. These polymers occur in nature as a storage reserve in some microbes faced with nutrient limitation (Madison et al., (1999) Microbiol. Mol. Biol. Rev. 63:21-53) and possess properties enabling their use in a variety of applications currently served by petroleum-based plastics. PHA biobased plastics can be produced via commercial large scale fermentations of microbial strains and the marketing of these plastics in a variety of applications is well under way (Bieles, (2006), Plastics and Rubber Weekly, Feb. 17:1; Deligio, (2007), Modern Plastics). Since they are inherently biodegradable in soil, compost, and marine environments, they can decrease plastic waste disposal issues. Pathways for production of PHAs have been introduced into a number of crops (for review, see Suriyamongkol et al., (2007), Biotechnol. Adv. 25:148-175 and references therein) including maize (Poirier et al., (2002), Biopolymers: Polyesters I—Biological Systems and Biotechnological Production (Doi Y and Steinbüchel A eds), pp. 401-435, Weinheim, Wiley-VCH), sugarcane (Petrasovits et al., (2007), Plant Biotechnol. 5:162-172; Purnell et al., (2007), Plant Biotechnol. 5:173′-184), flax (Wrobel-Kwiatkowska et al., (2007), Biotechnol. Prog. 23:269-277; Wrobel et al., (2004), J. Biotechnol. 107:41-54), cotton (John et al., (1996), Proc. Natl. Acad Sci. USA 93:12768-12773), alfalfa (Saruul et al., (2002), Crop Sci. 42:919-927), tobacco (Arai et al., (2001), Plant Biotechnol. 18:289-293; Bohmert et al., (2002), Plant Physiol. 128:1282-1290; Lössl et al., (2005), Plant Cell Physiol. 46:1462-1471; Lössl et al., (2003), Plant Cell Rep. 21:891-899), potato (Bohmert et al., (2002), Plant Physiol. 128:1282-1290), and oilseed rape (Houmiel et al., (1999), Planta 209:547-550; Slater et al., (1999), Nat. Biotechnol. 17:1011-1016; Valentin et al., (1999), Int. J. Biol. Macromol. 25:303-306) resulting in the production of a range of polymer levels depending on the crop and mode of transformation. See also U.S. Pat. No. 5,663,063 to Peoples et al., and U.S. Pat. No. 5,534,432 to Peoples et al. To date, significant PHA production has only been demonstrated in plants with a C3 photosynthetic pathway (i.e., Arabidopsis, Brassica) or a C4 NADP-malic enzyme photosynthetic pathway (corn, sugarcane) that produce storage products such as oils or carbohydrates. Production of polymer in plants with a C4 NAD-malic enzyme photosynthetic pathway such as switchgrass has not been demonstrated. Moreover, PHB production greater than 0.3% dwt (dry weight) has only been reported in plants that produce storage materials such as oils or carbohydrates. It is unknown whether plants that do not produce storage products can be engineered to produce PHAs in commercially useful amounts.
Switchgrass is one of the bioenergy crops targeted by the United States Department of Energy (DOE (2006), U.S. Department of Energy Office of Science and Office of Energy Efficiency and Renewable Energy (www.doegenomestolife.org/biofuels/); Sanderson et al., (2006), Can. J. Plant Sci. 86:1315-1325). Recent studies suggest that production of cellulosic ethanol from this crop nets 540% more renewable energy than the required nonrenewable energy inputs (Schmer, et al., (2008), Proc. Natl. Acad Sci USA 105:464-469). Despite the considerable interest in application of genomics and transgenic approaches for improvement of switchgrass for biofuel production (Bouton, (2007), Current Opinion in Genetics & Development 17:553-558), only the expression of reporter and selectable marker genes has been described (Richards et al., (2001), Plant Cell Rep. 20:48-54; Somleva et al., (2002), Crop Sci. 42:2080-2087). Additionally, switchgrass does not produce storage products such as oils or carbohydrates, and thus would not be expected to accumulate PHA.